TAILIEUCHUNG - Báo cáo khoa học: The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72lmcGMP for half-maximal effective concentration (EC50)ofa cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we exam-ine whether modifications in the N-terminus of PDE11A4 affect cGMP sig-nalling. | ỊFEBS Journal The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain Marco Gross-Langenhoff1 Arnulf Stenzl2 Florian Altenberend1 Anita Schultz1 and Joachim E. Schultz1 1 Pharmazeutisches Institut Universitat Tubingen Germany 2 Urologische Universitatsklinik Universitat Tubingen Germany Keywords adenyl cyclase cGMP cyclic nucleotide phosphodiesterase 11 GAF tandem domain regulation Correspondence J. E. Schultz Pharmazeutisches Institut Universitat Tubingen Morgenstelle 8 72076 Tubingen Germany Fax 49 7071 295952 Tel 49 7071 2974676 E-mail Received 22 October 2007 revised 4 December 2007 accepted 5 February 2008 doi The tandem GAF domain of human phosphodiesterase 11A4 hPDE11A4 requires 72 pM cGMP for half-maximal effective concentration EC50 of a cyanobacterial adenylyl cyclase used as a reporter enzyme. Here we examine whether modifications in the N-terminus of PDE11A4 affect cGMP signalling. The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases Ser117 Ser168 . Phosphorylation of both by cAMP-dependent protein kinase decreased the EC50 value for cGMP from 72 to 23 pM. Phosphomimetic point mutations S117D S167D which project complete phosphorylation lowered the EC50 value to 16 pM. Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4. Removal of 197 amino acids yielded unregulated cyclase activity whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC50 value of pM. Truncation by 176 amino acids was required for cGMP EC50 values to decrease to below 10 pM a construct truncated by 168 amino acids had an EC50 value of 224 pM. The decrease in EC50 values was accompanied by a sixfold increase in basal activity the extent of cGMP stimulation remained unaffected however. We conclude that N-ter-minal .

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